A red nucleus–VTA glutamate pathway underlies exercise reward and the therapeutic effect of exercise on cocaine use

Physical exercise is rewarding and protective against drug abuse and addiction. However, the neural mechanisms underlying these actions remain unclear. Here, we report that long-term wheel-running produced a more robust increase in c-fos expression in the red nucleus (RN) than in other brain regions. Anatomic and functional assays demonstrated that most RN magnocellular portion (RNm) neurons are glutamatergic. Wheel-running activates a subset of RNm glutamate neurons that project to ventral tegmental area (VTA) dopamine neurons. Optogenetic stimulation of this pathway was rewarding, as assessed by intracranial self-stimulation and conditioned place preference, whereas optical inhibition blocked wheel-running behavior. Running wheel access decreased cocaine self-administration and cocaine seeking during extinction. Last, optogenetic stimulation of the RNm-to-VTA glutamate pathway inhibited responding to cocaine. Together, these findings indicate that physical exercise activates a specific RNm-to-VTA glutamatergic pathway, producing exercise reward and reducing cocaine intake.


Figure S1 (
Figure S1 (related to Fig. 1): A series of images from the rostral to caudal levels of the RN/VTA.Wheel-running increased c-fos expression in the RNm relative to sedentary (wheellocked) mice.

Figure S2 (
Figure S2 (related to Fig. 1): Wheel running increases c-fos expression in a subset of VTA DA neurons.A: VTA c-fos expression in a sedentary control mouse when the wheel was locked; B: VTA c-fos expression in a wheel-running mouse; C: Quantitative cell counting indicates that ~10% of VTA DA neurons (marked by arrows) displayed c-fos expression after wheel-running.

Figure S3 (
Figure S3 (related to Fig. 2): Series of brain slice images, illustrating GABAergic vs. glutamatergic neuronal distributions from the rostral RNp to the caudal RNm.A: AAV-

Figure S4 (
Figure S4 (related to Fig. 3): Optical conditioned place preference (CPP) produced by laser stimulation of RN-VTA glutamate projection terminals in the VTA.A: A schematic diagram showing the general experimental methods.AAV-ChR2-eYPF was microinjected into bilateral RNm, while optical fibers were implanted to the VTA to stimulate ChR2-expressing glutamate terminals.B: The sequence of 15 days of CPP procedures; C: Three days of laser stimulation of RNm glutamate terminals in the VTA produced CPP in an additional group of mice, which lasted for 3 days.A one-way RM ANOVA analysis reveals significant laser treatment main effect (F4,24=3.85,p<0.05).A post-hoc Dunnett's test indicated that CPP scores on day 1 and day 3 were significantly higher than those on the pre-conditioning session.*p<0.05,**p<0.01,as compared to pre-conditioning.

Figure S5 (
Figure S5 (related to Fig. 4): Bidirectional neural projections between the RN and VTA.A: RNm-VTA glutamate projection as assessed by unilateral microinjection of AAV-ChR2-eYFP into the RNm.B: AAV-ChR2-eYFP was injected into bilateral RNm to express ChR2-eYFP in glutamate neurons.The eYFP-labeled fibers (green) project from the RN to or go through the contralateral VTA; C: The eYFP-labeled glutamatergic fibers also project from the RNm to the ipsilateral VTA; D: TH-positive DA neurons project from the VTA to the ipsilateral RNm.

Figure
Figure S6 (related to Fig. 4 and Fig. 5): Fluoro-gold (FG) retrograde tracing of RNm afferents.A: The classical retrograde tracer FG was microinjected into one side of the RNm.FGlabeled cells were detected in the ipsilateral SNr (substantia nigra pars reticulata), contralateral RNm, and bilateral superior colliculus; B/C: FG-labeled cells were also found in the ipsilateral cortex (possibly motor cortex) and bilateral cerebellum.

Figure S7 (
Figure S7 (related to Fig. 6): Optical stimulation of VTA DA neurons was both locomotorstimulating and rewarding.A: A schematic diagram showing that AAV-ChR2-eYFP was microinjected into bilateral VTA and the stimulation optrodes targeted bilateral VTA in DAT-cre mice; B/C: Laser stimulation of VTA DA neurons increased open-field locomotor activity (oneway RM ANOVA, F23,138 = 5.59, p<0.001);D: Laser stimulation of VTA DA neurons produced significant CPP in DAT-Cre mice (paired t-test, p<0.001);E: Laser stimulation of VTA DA neurons produced robust oICSS behavior as assessed by active lever responses over inactive lever responses; F: Representative oICSS responses (active lever presses) maintained by optical stimulation of VTA DA neurons (n=4).*p<0.05,***p<0.001,as compared to baseline before laser stimulation (C) or pre-conditioning (D).

Figure S9 (
Figure S9 (related to Fig. 10): Effects of optical inhibition of RNm glutamate neurons on cocaine self-administration.A: Schematic diagrams showing the general experimental methods.AAV-eYFP control virus was injected into the bilateral RNm, while the stimulation optrodes targeted the RNm; B: Optical stimulation of the RNm failed to alter cocaine self-administration in mice having received the AAV-eYFP microinjections; C: Representative cocaine selfadministration records, illustrating that laser stimulation of the RNm had no effect on cocaine selfadministration.D: Schematic diagrams showing the general experimental methods.AAV-NpHR-

Figure S10 (
Figure S10 (related to Fig. 10): Effects of optical stimulation of RNm glutamate terminals in the VTA on basal or cocaine-enhanced locomotion.A: A schematic diagram showing the experimental methods; B: Laser stimulation of RNm glutamate terminals failed to alter basal level of locomotion; C: Laser stimulation of RNm glutamate terminals in the VTA also failed to alter cocaine-induced hyperactivity.

Figure S11 (
Figure S11 (related to Fig. 10): Effects of optical manipulations of VTA glutamate neurons or RN GABA neurons on cocaine self-administration.A/B: Optical stimulation of glutamate neurons in the VTA failed to alter cocaine self-administration in VgluT2-Cre mice that had received AAV-ChR2-eYFP microinjections; C/D: Stimulation of GABA neurons in the RNm failed to alter cocaine self-administration in vGAT-Cre mice that had received AAV-ChR2-eYFP microinjections; E/F: Optical inhibition of GABA neurons in the RNm also failed to alter cocaine self-administration in vGAT-Cre mice that had received AAV-NpHR-eYFP microinjections.

Figure S12 (
Figure S12 (related to Fig. 10).Effects of optical stimulation of glutamate neurons in the vHipp on cocaine self-administration.A: Representative images showing c-fos expression in hippocampus in both wheel-running and wheel-locked mice.Wheel-running increased c-fos expression in the hippocampus.B: A schematic diagram showing the experimental methods; C: Representative images showing AAV-ChR2-eYFP expression in vHipp; D: laser stimulation of glutamate neurons in vHipp failed to alter cocaine self-administration in VgluT2-Cre mice.